1. Field of the Invention
The present invention relates to a process for the production of a physiologically active peptide containing at least one cysteine residue (hereinafter abbreviated as target peptide).
2. Description of the Related Art
There have been many attempts to obtain physiologically active peptides or proteins of eukaryote origin in microbial cells such as Escherichia coli cells using recombinant DNA techniques.
To produce a relatively small size peptide which is readily degraded in microbial cells such as E. coli, a process is usually used wherein a desired peptide or protein is produced in microbial cells as a protein fusion with another peptide or protein, the produced fusion protein is then chemically or enzymatically cleaved to liberate the desired peptide or protein, and the desired peptide and protein is isolated and purified.
Various methods are known for liberating a desired peptide from a fusion protein. In the case of a desired peptide not containing a methionine residue, the desired peptide is produced as a fusion protein wherein the desired peptide is fused with a partner protein via a methionine residue, and the desired peptide is liberated from the partner protein by cleavage at the methionine residue with cyanogen bromide (Science, 198, 1059 (1977); and Proc. Natl. Acad. Sci. USA, 76, 106 (1978)). In the case of a desired peptide not containing an arginine or lysine residue, the desired peptide is produced as a fusion protein wherein the desired peptide is fused with a partner protein via the arginine or lysine residue, and the desired peptide is liberated from the partner protein by proteolytic cleavage at the arginine or lysine residue with trypsin, which specifically cleaves a peptide bond at the C-terminal of the arginine or lysine residue (Nature, 285, 456, 1980). Moreover, in some cases, Achromobacter protease I (hereinafter abbreviated as API, also known as lysyl endopeptidase), which specifically cleaves a peptide bond at the C-terminal of a lysine residue, may be used.
In many cases, a partner protein which forms a fusion protein in combination with a desired peptide is selected from a protein which is naturally produced by a host organism. In this case, usually, a polypeptide fragment having an appropriate size derived from the N-terminal part of a protein which is produced in large amounts by the host is used as a partner protein. In such a case, it is considered that the size of the partner protein affects the productivity of the desired peptide. For example, although it is thought that by using a smaller size of a partner protein a larger amount of a desired peptide is obtained, because of a high proportion of the desired peptide in the fusion protein. This does not always occur. For example, in the case of production of insulin using E. coli .beta.-galactosidase as a partner in a fusion protein, although a decrease of the size of the .beta.-galactosidase region provides an increase in the production of the target insulin, a further decrease of the .beta.-galactosidase size decreases the production of the target insulin (Gene, 29, 251, 1984). As seen from the above, when a particular peptide is to be produced as a fusion protein, there is no rule determining a size of a partner protein which provides maximum productivity of a desired peptide.
Therefore, the most important point for efficient production of a target peptide is the selection of the most suitable partner protein for the target peptide efficiently to express a fusion protein of the target peptide and the partner protein, and efficiently recover the target peptide from the fusion protein. However, since a general rule for such a selection has not been established, the optimum conditions for a particular target peptide must be found by experiment.
Next, as examples of a target peptide containing cysteine residues, .alpha.-type human atrial natriuretic polypeptide (hereinafter abbreviated as .alpha.-hANP), and human calcitonin precursor (hereinafter abbreviated as HPTC) are mentioned, and the problems in the production of the these peptides by the recombinant DNA technique are described in detail.
The .alpha.-hANP is a peptide consisting of 28 amino acid residues, containing two cysteine residues at portions 7 and 23 which form an intramolecular disulfide bond, and having the following formula (I): ##STR1## wherein (1) and (2) are directly bonded.
The .alpha.-hANP was extracted and purified from human atria by Matsuo and Kangawa (European Patent Publication No. 0147193). The .alpha.-hANP exhibits a notable natriuretic activity and blood pressure lowering activity (Biochem. Biophys. Res. Commun. 118, 131-139, 1984).
On the other hand, the HPCT is a peptide consisting of 36 amino acid residues, containing two cysteine residues at positions 1 and 7 which form an intramolecular disulfide bond, and having the following formula (II): ##STR2## wherein (1) is directly bonded with (2), and (3) is directly bonded with (4).
The HPCT is known as an intermediate of human calcitonin corresponding to a sequence from the first amino acid to the 32nd amino acid in the formula (II) (Nature 295 345-347, 1982).
Although these peptides are now chemically synthesized, the process for chemical synthesis of a large amount of these peptide is labor intensive and time-consuming. Therefore, since these peptide in particular are intended for use as pharmaceuticals, a simple and inexpensive process for large scale production of these peptides is strongly desired.
To resolve the above-mentioned problems, attempts have been made to produce such peptides using a recombinant DNA technique. For example, to produce .alpha.-hANP, .alpha.-hANP is expressed as a fusion protein comprising the .alpha.-hANP and E. coli Trp E protein, and a crude extract containing the fusion protein prepared from a cell disruptant is treated with lysyl endopeptidase or a coagulation factor Xa to liberate the .alpha.-hANP from the fusion protein (Seikagaku, 57(8), 854(1984). In this procedure, however, the recovery process is complicated and the recovery ratio of the target peptide from the fusion protein is low, and thereofor, this procedure is not practical. To resolve the problem, the present inventors invented a process to produce a physiologically active peptide not containing lysine residues represented by .alpha.-hANP. The target peptide is expressed as a fusion protein comprising the target peptide and a partner polypeptide of 90 to 220 amino acid residues not containing lysine residues linked via a lysine residue, and the fusion protein is treated with lysyl endopeptidase to liberate the target peptide (Japanese Patent Application No. 61- 101100). However, this improved process does not provide a satisfactory recovery efficiency of the target peptide.
On the other hand, for the production of the HPCT, the present inventors reported a process for the production of a derivative of HPCT wherein the eighth methionine residue is converted to valine residue in the formula (II) (Japanese Patent Application No. 58-203953). In this process, the target peptide is expressed as a fusion peptide with E. coli alkaline phosphatase, and then the fusion protein is treated by cyanogen bromide to obtain the target peptide. However, this process, as described for .alpha.-hANP, also does not have a satisfactory recovery efficiency of the target peptide from the fusion protein. In contrast to these cases, it is known that, in the production of a peptide not containing cysteine residues by a recombinant DNA technique, the recovery efficiency of the target peptide from fusion protein is higher than the case of a target peptide containing cysteine residue(s). Therefore, a key factor in the efficient production of a peptide containing cysteine residue(s) is an increase of the recovery efficiency of the target peptide from the fusion protein. A solution to this problem is strongly desired at this time.
Therefore, the present invention is intended to provide a process which can be applied to a large scale production of a peptide containing cysteine residue(s), and especially, relates to the selection of a partner protein and a linker amino acid which bond the partner protein and the target peptide in fusion protein.